The actions of the intracellular messengers inositol trisphosphate (InsP3) and calcium ions will be investigated in invertebrate photoreceptors. Illumination of invertebrate photoreceptors produces InsP3 via the phosphoinositide (PI) pathway of signal transduction. Molecular components of the PI pathway have been identified within invertebrate photoreceptors, but a complete explanation of their role in transduction has not yet emerged. Although the PI pathway is unlikely to play a role in excitation of vertebrate photoreceptors, a complete understanding of invertebrate phototransduction can be applied to other cells that respond to hormones or neurotransmitters which activate the PI pathway, including iris smooth muscle cells and retinal neurons. In Limulus (horseshoe crab) photoreceptors, InsP3 releases calcium ions from endoplasmic reticulum. The released calcium ions "mimic" the effect of light by opening sodium channels in the plasma membrane. Novel InsP3 isomers will be used to demonstrate that the response to InsP3 is mediated by stereospecific receptor site. We will continue to examine feedback inhibition by calcium ions of InsP3-induced calcium release, in order to demonstrate its rapid reversibility. Feedback control of calcium release may play a role in lowering intracellular calcium ion concentration (Cai) during stimulation of mammalian cells, preventing cell injury and death during ischemia. Metabolism of InsP3 will also be investigated using novel InsP3 analogues. Prior injection of a novel inhibitor of the enzymes that destroy InsP3, l-chiro 1,4,6,InsP3, causes single injections of InsP3 to produce sustained oscillatory bursts of calcium release. These bursts are not normal components of the light response. The inhibitor will be used to determine whether rapid destruction of InsP3 after a light flash prevents their occurrence. Inhibition of InsP3 metabolism by analogues should have general applications, since there are few drugs presently targeted against the PI pathway. Lithium's anti-manic effects may result from inhibition of inositol phosphate metabolism. A second set of experiments will determine whether the InsP3-induced elevation of (Cai) is necessary for visual excitation or whether another pathway for excitation must exist. We will use fluorescent calcium indicators to determine whether excitation of Limulus photoreceptors by light can occur in the absence of detectable elevation of Cai. We will measure Cai during the collapse of the light response of mutant fly photoreceptors (nss Calliphora), which are unable to sustain a response to bright illumination.